Surveys of human genomic DNA have indicated that tandemly reiterated sequences are present in abundance (Stallings, Genomics, 1994. 21: p. 116-21; Han et al., Nucleic Acids Res.,1994. 22(9): p. 1735-40). The polymorphic nature of these sequences has fostered their use in a variety of studies. Recently a number of human diseases have been shown to be caused by the expansion of a subset of these repetitive sequences, trinucleotide repeats (HDCRG, Cell, 1993, 72(6): p. 971-83; Fu et al., Science, 1992. 255(5049): p. 1256-8; Knight et al., Cell, 1993, 74(1): p. 127-34; Orr et al., Nat Genet, 1993, 4(3): p. 221-6; Harley et al., Nature, 1992, 355(6360): p. 545-6; Buxton et al., Nature, 1992, 355(6360): p. 547-8; Aslanidis et al., Nature, 1992, 355(6360): p. 548-51; La-Spada et al., Nature, 1991. 352(6330): p. 77-9; Sutherland et al., Lancet, 1991, 338(8762): p. 289-92; Yu et al., Science, 1991, 252(5010): p. 1179-81; Kremer et al., Science, 1991. 252(5013): p. 1711-4; Verkerk et al., Cell, 1991, 65(5): p. 905-14; Koide et al., Nat Genet, 1994, 6(1): p. 9-13).
All of the currently known diseases caused by trinucleotide repeats are caused by repeats high in dG+dC (guanine and cytosine respectively) content (Han et al., 1994). One method for analyzing the expansion of such repeats is by amplifying the region using the polymerase chain reaction (PCR). The high dG+dC content renders amplification and/or DNA sequencing very difficult due to an increased melting temperature, or T.sub.m, and stable secondary structure of the expanded motif. A common result of amplifying a region containing a repeat motif with a high dG+dC content is the presence of additional amplification products which do not correspond to the desired product (Hauge et al., Hum. Molec. Genet., 1993, 2(4): p. 411-15). Such "stutter" or "shadow" banding complicates the interpretation of results of an assay. A number of authors have noted the difficulty in interpreting the banding patterns seen in Huntington's disease (HD) (Riess, O., et al., Hum Mol Genet, 1993, 2(6): p. 637; Goldberg et al., Hum Mol Genet, 1993. 2(6): p. 635-6; Valdes et al., Hum Mol Genet, 1993, 2(6): p. 633-4; Snell et al., Nat Genet, 1993, 4(4): p. 393-7; Barron et al., Hum. Molec. Genet., 1994, 3(1): p. 173-175).
Several theories addressing the problem of "stutter" or "shadow" banding have been put forth (Litt et al., Biotech., 1993, 15(2): p. 280-284). Possible mechanisms resulting in false banding patterns may include improper primer annealing to a repetitive sequence or strand slippage during synthesis. A third explanation proposes that secondary structure unique to the repetitive sequences allow the extending DNA strand to skip cassettes of repeats. If this were to occur during the early cycles of a PCR reaction sufficient template could be made which would eventually appear as additional or "stutter" bands. Secondary structure resulting in additional banding may be caused by the increased stability of a region with an increased dG+dC content. The differential stability of base pairs has been a subject of inquiry for over three decades. Phosphate binding cations have long been known to be general destabilizers of the DNA helix (von Hippel et al., Ann. Rev. Biochem., 1972, 41: p. 231-300) The most likely mechanism for this alteration of helical stability is the affect that these cations (Cs.sup.+, Li.sup.+, Na.sup.+, K.sup.+, Rb.sup.+, Mg.sup.++, Ca.sup.++) have on the transfer of free energy of a nucleotide from a non-aqueous to an aqueous environment (von Hippel et al., 1972). These cations effectively increase the solubility of nucleotides in aqueous solutions which acts to destabilize the helix in a general fashion.
Another class of compounds has been shown to alter relative stability of the DNA helix based on nucleotide composition. Various tetraalkylammonium ions are known to preferentially bind in DNA grooves at dA.dT base pairs (Melchior et al., PNAS, 1973, 70(2): p. 298-302). The mechanism in this case relies on the differential levels of hydration between base pairs and the size of the tetraalkylammonium ion being used. Previous work has suggested that dA.dT base pairs are more highly hydrated than dG.dC base pairs thus providing a relatively more suitable binding site for the nonpolar arms of alkylammonium ions (Tunis et al., Biopolymers, 1968, 6: p. 1218-1223). It has also been demonstrated that larger tetraalkylammonium ions are general destabilizers of DNA while smaller tetraalkylammonium ions have a differential stabilization effect based on base composition (Melchior et al., 1973). The overall effect, in this case, is to produce a relative isostabilization of the dA.dT base pairs relative to dG.dC base pairs thus eliminating the base composition contribution to the T.sub.m of a DNA sequence. Isostabilization is desirable in determining a T.sub.m at which DNA secondary structure would be minimal. The use, however, of tetraalkylammonium compounds in these studies is offset by their destabilization effect on DNA-protein interactions at the salt concentrations necessary to achieve DNA isostabilization (Rees et al., Biochemistry, 1993, 32(1): p. 137-44).
There is a need for a compound which would offer the isostabilizing effect of the tetraalkylammonium compounds without the DNA-protein altering side effects.